Regulation of gene expression in eukaryotes; lecture 20

Promoters

Promoters, which bind RNA polymerase II There are only a few types of promoters and promoter proximal elements. The most common promoter element is the TATA box, which binds TBP. The transcription factors that bind to promoters or promoter proximal elements are usually ubiquitously expressed and affect many different genes.

transcription factors

Proteins that bind to eukaryotic regulatory sequences

How can eukaryotic transcription factors control gene transcription?

Similar to bacteria, but more complicated - Direct interaction / interference with RNA pol II - Indirect interaction with RNA pol II (via bridging proteins) - Alteration of chromatin state

Enhancer (Enhancer) & Silencer (Silencer)

Enhancers and silencers, which bind proteins that regulate gene expression through several possible mechanisms. Enhancers and silencers are regulatory elements that can control the activity of a promoter even at a great distance (up to 100 kb!). Enhancers/silencers can be located either upstream or downstream of the promoter (in the gene, downstream of the gene, pretty much wherever). *Enhancers increase transcription rates.* *Silencers reduce transcription*. Enhancers and silencers are usually responsible for directing the expression of a gene to certain cell types or certain developmental periods. Individual enhancers will often direct expression of a gene only in a organ or tissue or at a specific time. *Combination of multiple enhancers will lead to the overall expression pattern of the gene.*

Histone acetyl-transferase, HAT (Histon-Acetyl-Transferase) & Histone deacetylase, HDAc (Histon-Deacetylase)

Acetylation of histones results in the opening of the nucleosomal structure (possibly through charge neutralization). - Acetylated histones = active chromatin - Deacetylated histones = inactive chromatin Acetylation is reversible - Acetylation: Histone acetyl-transferase (*HAT*) - Deacetylation: Histone deacetylase (*HDAc*)

X-chromosome inactivation

During early embryonic development, each cell in a female embryo randomly inactivates ("shuts down") one of its two X chromosomes. The inactivated chromosome becomes heterochromatin (visible as the Barr body in microscopy). The X chromosome inactivation pattern is stably inherited to all daughter cells of that embryonic cell: the inactivated chromosome stays inactivated, the active chromosome stays activated. Mammalian females are thus functionally mosaics: they have some patches of cells that express genes from only one X chromosome, and other patches that express genes only from the other chromosome.

Chromatin remodeling

Even in open chromatin, a promoter might be inaccessible to RNA pol II if the TATA box happens to be tightly bound to a nucleosome. Chromatin remodeling complexes have the capacity to displace individual nucleosomes. This can move the TATA box into a free DNA region, where it can then be bound by TBP.

Epigenetics (Epigenetik)

Heritable modification in gene function / gene expression that is not due to changes in the base sequence in the DNA. Epigenetic states can be inherited through mitosis, and in some cases even through meiosis (i.e., from one generation to the next). Epigenetic inheritance can be due to stable association of protein with a region of DNA, or the stable modification of this DNA (e.g. methylation). Genome (Nature) + Environment (Nurture) = Phenotype

Acetylation

Histones are rich in positively charged amino acids, which neutralize the negative charge of the phosphates in DNA. Lysine acetyl-transferases (KATs) can transfer an activated acetic acid molecule (usually from acetyl-CoA) to the the amine on the ε carbon of lysine, to create an *amide bond*. Acetylation of lysine neutralizes the positive charge of the side chain (amides are not charged). Acetylation occurs on lysines residues, and is sequence specific.

Dosage compensation (Dosis Kompensation)

Humans have 22 pairs of autosomes, and 1 pair of sex chromosomes - XX in women - XY in males • Men and women thus have different numbers of X chromosomes (women have one too many or men have one too few, depending on your point of view...). • Changes in chromosome numbers can result in very severe developmental defects. In humans, trisomy of only 3 autosomes is compatible with life, and monosomy is not allowed at all (see lecture 10). • So why is it that having different numbers of X and Y chromosomes is allowed? - There are very few Y chromosome-specific genes, and they are all only required in males. Proper expression of sex-linked genes is obtained through a process known as dosage compensation, which, by one mechanism or the other, insures that genes on sex chromosomes are expressed at similar levels in males and females. The mechanisms of dosage compensation are varied: - Mammals: X chromosomes inactivation - Drosophila: 2-fold increased expression of the single X chromosome in males - C. elegans: 2-fold decreased expression of the two X chromsomes in hermaphrodites.

Chromatin condensation

In active DNA, individual nucleosomes are separated by short strectches of free "linker" DNA, forming a "*beads on a string*" arrangement. In inactive DNA, nucleosomes are wrapped up into progressively larger and more condensed *solenoids*. The simplest of these is the 30 nm fiber. Chromatin is tightly attached to a protein scaffold, creating large regions that can be controlled independently of each other Specific regions, known as *scaffold attachment regions* (SARs), mediate attachment of the DNA to the scaffold. The chromatin structure of each loop can be controlled separately. SARs often thought to act as *insulators*, which restrict the effect of enhancers to promoters that are part of the same loop.

Marsupials (Beuteltiere)

In all mammals, the paternal X chromosome is preferentially inactivated in the extra-embryonic tissue. In marsupials, the paternal X chromosome is also preferentially inactivated in the embryo. Moreover, X inactivation is often incomplete in marsupials. In parental imprinting, certain autosomal genes are silenced in the germ line in a gender-specific manner. Some autosomal genes (e.g., igf2) are only expressed if they are on the chromosome inherited from the father. Other genes show the opposite phenotype. In the germ line, all imprinting marks are removed from both parental copies, and the proper "gender pattern" is put on both chromosomes (which will be transmitted with this "gender pattern" to the next generation).

DNA methylation (DNA Methylierung)

In mammals and plants, Cytosine residues in a CG dimer sequence are often methylated on C5 in heterochromatic regions. 5' ... mC G... 3' 3' ... G mC... 5' The 5-methyl modification of C does not affect its base pairing with G, but can be recognized by proteins that bind to methylated DNA and recruit HDAcs, thereby insuring continued repression of gene expression. DNA methylation patterns can easily be reestablished after DNA replication through the action of a maintenance DNA methylase. (viel stabiler als Acetylisierung) DNA methylation is less dynamic than histone modification, and is usually only found in stably repressed heterochromatin. Nucleosomes disassemble during DNA replication; new nucleosomes carrying both old and new histones are reloaded onto both daughter strands. Modification enzymes soon reestablish the original modification pattern, most likely by using the modifications on the old histones as a guide. *Genes that were active in the mother cell will thus be allowed to be active in the daughter cells. Genes that were silenced in the mother stay silenced in the daughters.*

Regulation by Hat & HDac

Many activator proteins either possess a HAT activity, or recruit a HAT once they are bound to enhancers. The HAT acetylates the histones around the enhancer, resulting in an open chromatin structure. Repressors can recruit HDAc complexes to silencers. The HDAc deactylates the surrounding histones, allowing the chromatin to condense.

Promoter proximal element (Promotor-proximales Element)

Promoter-proximal elements (upstream promoter elements, UPEs), which bind proteins that assist binding of RNA pol II. UPEs are located 100-200bp upstream of the transcription start site. Common promoter proximal elements include the CAAT box and the GC box Promoter proximal elements rarely determine where a gene is expressed, but rather how strongly it is expressed.

Histone tails

The N- and C-termini ("tails") of all 8 core histones peek out of the nucleosome complex, and are accessible to modifying enzymes. Modification of histones alters chromatin structure, and likely can also send other messages (concept of the *histone code*). Histone tails can be subject to many different modifications: - Acetylation - Methylation (mono, di, tri) - Phosphorylation - Ubiquitylation

DNA wrapping

The basic unit in the chromatin is the nucleosome. Nucleosomes have a core consisting of 8 histone protein molecules - 2x H2A - 2x H2B - 2x H3 - 2x H4 DNA makes two turns around each nucleosomes = 146 bp In condensed chromatin, the DNA between nucleosomes is bound to histone H1. Eukaryotic DNA is tightly packaged into chromatin. Eukaryotic transcription factors will thus use several different mechanisms to promote gene transcription: - Direct interaction with RNA pol II - Indirect interaction with RNA pol II (via coactivators) - Chromatin remodeling - Modulation of chromatin condensation

Heterochromatin

condensed chromatin. Genes in heterochromatin regions are usually silenced (not expressed). - Constitutive heterochromatin: always condensed. - Facultative heterochromatin: Regions that can be open under some conditions (e.g. in some cell types, or during some developmental stages), and condensed under others.

Euchromatin

open chromatin. Genes in euchromatin regions can be transcribed (if the right transcription factors are present).

Summary

• In eukaryotes, transcription is regulated by sequences that can either be close to the promoter (promoter proximal elements), or sequences that can be very far away (enhancers and silencers). • Combinatorial and cooperative binding of regulatory proteins to regulatory sequences allows a limited number of transcription factors to generate a very wide range of expression patterns. • Regulation of chromatin structure is an essential component of the regulation of transcription in eukaryotes. • Chromatin remodeling complexes can move individual nucleosomes. • Euchromatin (active) and heterochromatin (inactive) can be distinguished by their DNA methylation pattern and by their histone modification pattern. • Active and inactive chromatin states can be inherited. This is known as epigenetic inheritance. • Example of epigenetic inheritance in mammals include X-inactivation and parental imprinting.

Chromatin condensation regulation

• Modification of core histones • Methylation of DNA • Stable association with specific proteins